Emergence of antimicrobial resistance is becoming a very large public health threat and has been recognized by, amongst others, the World Health Organization, the U.S. Congress Office of Technology Assessment and the United Kingdom House of Lords. The urgency to develop new classes of antimicrobial agents particularly against Gram-negative pathogens Acinetobacter baumannii and Pseudomonas aeruginosa, was demonstrated by the dramatic increases in the incidence of antibiotic-resistant species in a recent study in Mexico. In one study, 550 clinical isolates of A. baumannii and 250 clinical isolates of P. aeruginosa were analyzed for the prevalence of multi-drug resistance, and 74% of A. baumannii and 34% of P. aeruginosa were multi-drug resistant.
Antimicrobial peptides (AMPs) are widely distributed in nature and represent a promising class of new antimicrobial agents. AMPs are rapidly bactericidal and generally have broad-spectrum activity. It is difficult for bacteria to develop resistance to AMPs because their mode of action involves nonspecific interactions with the cytoplasmic membrane. In addition, enantiomeric forms of AMPs with all-D-amino acids have shown equal activities to their all-L-enantiomers, suggesting that the antimicrobial mechanism of such peptides does not involve a stereoselective interaction with a chiral enzyme or lipid or protein receptor. In addition, all D-peptides are resistant to proteolytic enzyme degradation, which enhances their potential as therapeutic agents. However, it is widely believed that native AMPs lack specificity and might be too toxic (including due to the ability to lyse mammalian cells, normally expressed as hemolytic activity against human red blood cells) to be used for systemic treatment.
The Dermaseptins are a family of linear peptides, initially isolated in 1991 from the skin of various tree-dwelling, South American frogs of the Phyllomedusa species. These amphipathic α-helical cationic antimicrobial family of peptides are structurally and functionally related. They exhibit rapid cytolytic activity against a variety of microorganisms including viruses, bacteria, protozoa, yeast and filamentous fungi. Unlike other Dermaseptin members, Dermaseptin S4, a 28-residue AMP, lyses erythrocytes at micromolar concentrations. (The Dermaseptin S4 was originally identified as a 28-mer peptide, but a deletion of a single residue in the sequence by the inventors produced an active 27-mer. For accuracy, the two peptides are referred to herein as “Dermaseptin S4, 27-mer”, and “Dermaseptin S4, 28-mer.” The sequences of these peptides are shown in Table 5 and additional data regarding the two forms is shown in FIG. 3.) The HC50, the peptide concentration that causes 50% lysis of human red blood cells, was approximately 1.4 μM using a three hour incubation time at 37° C. Very rapid kinetics (within seconds) for the lysis of human red blood cells can be observed under a microscope. This hemolytic activity of these peptides is a significant disadvantage for the therapeutic use of these peptides.
The Piscidin family comprises the most common group of AMPs in teleost fish. Piscidin 1 was first isolated in 2001 from hybrid striped bass (Morone saxatilis male×Morone chrysops female), where it is produced in mast cells (immune cells of uncertain function present in all vertebrates), skin, gill and gastrointestinal tract. Piscidin 1 is a 22-residue amphipathic α-helical AMP rich in histidines and phenylalanines. Piscidin 1 has the highest biological activity among the family with broad-spectrum activity against antibiotic-resistant bacteria, filamentous fungi, yeasts, and viruses. This peptide, however, is not selective for bacterial versus mammalian cells, and caused hemolysis of human red blood cells with a HC50 of 11˜20 μM within one hour at 37° C. Thus, both AMPs from different natural sources suffer the dose or drug-limiting toxicity of hemolytic activity.
Thus, a new class of antimicrobial agents with lower rates of resistance and different microbial targets is urgently needed because of the rapidly increasing resistance to classical antibiotics. Amphipathic cationic α-helical antimicrobial peptides (AMPs) represent such a class of compounds, but the toxicity of these compounds to mammalian cells must be overcome.